Abstract
Liver sinusoidal endothelial cells (LSECs) represent a highly differentiated cell type that lines hepatic sinusoids. LSECs form a discontinuous endothelium due to fenestrations under physiological conditions, which are reduced upon chronic liver injury. Cultivation of rodent LSECs associates with a rapid onset of stress-induced senescence a few days post isolation, which limits genetic and biochemical studies ex vivo. Here we show the establishment of LSECs isolated from p19ARF-/- mice which undergo more than 50 cell doublings in the absence of senescence. Isolated p19ARF-/- LSECs display a cobblestone-like morphology and show the ability of tube formation. Analysis of DNA content revealed a stable diploid phenotype after long-term passaging without a gain of aneuploidy. Notably, p19ARF-/- LSECs express the endothelial markers CD31, vascular endothelial growth factor receptor (VEGFR)-2, VE-cadherin, von Willebrand factor, stabilin-2 and CD146 suggesting that these cells harbor and maintain an endothelial phenotype. In line, treatment with small molecule inhibitors against VEGFR-2 caused cell death, demonstrating the sustained ability of p19ARF-/- LSECs to respond to anti-angiogenic therapeutics. From these data we conclude that loss of p19ARF overcomes senescence of LSECs, allowing immortalization of cells without losing endothelial characteristics. Thus, p19ARF-/- LSECs provide a novel cellular model to study endothelial cell biology.
Highlights
Endothelial cells (ECs) represent a unique cell population originating from the mesoderm and lining all vessels in the organism
Loss of p19ARF allows proliferation of mouse p19ARF-/- liver sinusoidal endothelial cell (mLSEC) mLSECs were isolated from livers of female p19ARF-/- mice
Isolated mLSECs have a typical cobblestone-like morphology, and in comparison with Liver sinusoidal endothelial cells (LSECs) isolated from wt mice, mLSECs start to proliferate in vitro a few days after introduction into cell culture
Summary
Endothelial cells (ECs) represent a unique cell population originating from the mesoderm and lining all vessels in the organism. From large vessels to small capillaries, ECs form compact monolayers with semi-permeable properties, mediating transport of small metabolites, migration of immune cells and vessel tone [1, 2]. In comparison with other ECs, they possess a unique morphology due to multiple membranous pores, called fenestrations, which are organized into sieve plates [4]. Due to their size of about 0.2 μm, fenestrations mediate fast transfer of small molecules, which accelerates metabolic exchange under controlled selection
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have